The structure of the GM-CSF receptor complex reveals a distinct mode of cytokine receptor activation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific alpha subunit and a betac subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

Targeting sphingosine kinase 1 induces MCL1-dependent cell death in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy where despite improvements in conventional chemotherapy and bone marrow transplantation, overall survival remains poor. Sphingosine kinase 1 (SPHK1) generates the bioactive lipid sphingosine 1-phosphate (S1P) and has established roles in tumor initiation, progression, and chemotherapy resistance in a wide range of cancers. The role and targeting of SPHK1 in primary AML, however, has not been previously investigated. Here we show that SPHK1 is overexpressed and constitutively activated in primary AML patient blasts but not in normal mononuclear cells. Subsequent targeting of SPHK1 induced caspase-dependent cell death in AML cell lines, primary AML patient blasts, and isolated AML patient leukemic progenitor/stem cells, with negligible effects on normal bone marrow CD34+ progenitors from healthy donors. Furthermore, administration of SPHK1 inhibitors to orthotopic AML patient-derived xenografts reduced tumor burden and prolonged overall survival without affecting murine hematopoiesis. SPHK1 inhibition was associated with reduced survival signaling from S1P receptor 2, resulting in selective downregulation of the prosurvival protein MCL1. Subsequent analysis showed that the combination of BH3 mimetics with either SPHK1 inhibition or S1P receptor 2 antagonism triggered synergistic AML cell death. These results support the notion that SPHK1 is a bona fide therapeutic target for the treatment of AML.

Monoclonal antibody targeting of IL-3 receptor α with CSL362 effectively depletes CML progenitor and stem cells.

Despite the remarkable efficacy of tyrosine kinase inhibitors (TKIs) in eliminating differentiated chronic myeloid leukemia (CML) cells, recent evidence suggests that leukemic stem and progenitor cells (LSPCs) persist long term, which may be partly attributable to cytokine-mediated resistance. We evaluated the expression of the interleukin 3 (IL-3) receptor α subunit (CD123), an established marker of acute myeloid leukemia stem cells, on CML LSPCs and the potential of targeting those cells with the humanized anti-CD123 monoclonal antibody CSL362. Compared with normal donors, CD123 expression was higher in CD34(+)/CD38(-) cells of both chronic phase and blast crisis CML patients, with levels increasing upon disease progression. CSL362 effectively targeted CML LSPCs by selective antibody-dependent cell-mediated cytotoxicity (ADCC)-facilitated lysis of CD123(+) cells and reduced leukemic engraftment in mice. Importantly, not only were healthy donor allogeneic natural killer (NK) cells able to mount an effective CSL362-mediated ADCC response, but so were CML patients' autologous NK cells. In addition, CSL362 also neutralized IL-3-mediated rescue of TKI-induced cell death. Notably, combination of TKI- and CSL362-induced ADCC caused even greater reduction of CML progenitors and further augmented their preferential elimination over normal hematopoietic stem and progenitor cells. Thus, our data support the further evaluation of CSL362 therapy in CML.

Neuropilin-2 genomic elements drive cre recombinase expression in primitive blood, vascular and neuronal lineages.

We have established a novel Cre mouse line, using genomic elements encompassing the Nrp2 locus, present within a bacterial artificial chromosome clone. By crossing this Cre driver line to R26R LacZ reporter mice, we have documented the temporal expression and lineage traced tissues in which Cre is expressed. Nrp2-Cre drives expression in primitive blood cells arising from the yolk sac, venous and lymphatic endothelial cells, peripheral sensory ganglia, and the lung bud. This mouse line will provide a new tool to researchers wishing to study the development of various tissues and organs in which this Cre driver is expressed, as well as allow tissue-specific knockout of genes of interest to study protein function. This work also presents the first evidence for expression of Nrp2 protein in a mesodermal progenitor with restricted hematopoietic potential, which will significantly advance the study of primitive erythropoiesis. genesis 53:709-717, 2015. © 2015 Wiley Periodicals, Inc.

14-3-3ε and ζ regulate neurogenesis and differentiation of neuronal progenitor cells in the developing brain.

During brain development, neural progenitor cells proliferate and differentiate into neural precursors. These neural precursors migrate along the radial glial processes and localize at their final destination in the cortex. Numerous reports have revealed that 14-3-3 proteins are involved in many neuronal activities, although their functions in neurogenesis remain unclear. Here, using 14-3-3ε/ζ double knock-out mice, we found that 14-3-3 proteins are important for proliferation and differentiation of neural progenitor cells in the cortex, resulting in neuronal migration defects and seizures. 14-3-3 deficiency resulted in the increase of δ-catenin and the decrease of ß-catenin and αN-catenin. 14-3-3 proteins regulated neuronal differentiation into neurons via direct interactions with phosphorylated δ-catenin to promote F-actin formation through a catenin/Rho GTPase/Limk1/cofilin signaling pathway. Conversely, neuronal migration defects seen in the double knock-out mice were restored by phosphomimic Ndel1 mutants, but not δ-catenin. Our findings provide new evidence that 14-3-3 proteins play important roles in neurogenesis and neuronal migration via the regulation of distinct signaling cascades.

Efficacy of an Fc-modified anti-CD123 antibody (CSL362) combined with chemotherapy in xenograft models of acute myelogenous leukemia in immunodeficient mice.

The prognosis of older patients with acute myelogenous leukemia is generally poor. The interleukin-3 receptor α-chain (CD123) is highly expressed on the surface of acute leukemia cells compared with normal hematopoietic stem cells. CSL362 is a fully humanized, CD123-neutralizing monoclonal antibody containing a modified Fc structure, which enhances human natural killer cell antibody-dependent cell-mediated cytotoxicity. Six continuous acute myelogenous leukemia xenografts established from patient explants and characterized by cell and molecular criteria, produced progressively lethal disease 42-202 days after transplantation. CSL362 alone reduced engraftment of one of four and three of four acute myelogenous leukemia xenografts in the bone marrow and peripheral organs, respectively. A cytarabine and daunorubicin regimen was optimized using this model to identify potentially synergistic interactions with CSL362. Cytarabine/daunorubicin improved the survival of mice engrafted with four of four acute myelogenous leukemia xenografts by 31-41 days. Moreover, CSL362 extended the survival of cytarabine/daunorubicin-treated mice for two of two acute myelogenous leukemia xenografts, while augmentation of natural killer cell-deficient NSG mice with adoptively transferred human natural killer cells improved survival against a single xenograft. Interestingly, this enhanced CSL362 efficacy was lost in the absence of chemotherapy. This study shows that acute myelogenous leukemia xenografts provide a platform for the evaluation of new therapeutics, simulating complex in vivo interactions, and that the in vivo efficacy of CSL362 supports continued clinical development of this drug.

Immune insufficiency during GVHD is due to defective antigen presentation within dendritic cell subsets.

Alloreactivity after transplantation is associated with profound immune suppression, and consequent opportunistic infection results in high morbidity and mortality. This immune suppression is most profound during GVHD after bone marrow transplantation where an inflammatory cytokine storm dominates. Contrary to current dogma, which avers that this is a T-cell defect, we demonstrate that the impairment lies within conventional dendritic cells (cDCs). Significantly, exogenous antigens can only be presented by the CD8(-) cDC subset after bone marrow transplantation, and inflammation during GVHD specifically renders the MHC class II presentation pathway in this population incompetent. In contrast, both classic and cross-presentation within MHC class I remain largely intact. Importantly, this defect in antigen processing can be partially reversed by TNF inhibition or the adoptive transfer of donor cDCs generated in the absence of inflammation.

The GM-CSF receptor family: mechanism of activation and implications for disease.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pluripotent cytokine produced by many cells in the body, which regulates normal and malignant hemopoiesis as well as innate and adaptive immunity. GM-CSF assembles and activates its heterodimeric receptor complex on the surface of myeloid cells, initiating multiple signaling pathways that control key functions such as cell survival, cell proliferation, and functional activation. Understanding the molecular composition of these pathways, the interaction of the various components as well as the kinetics and dose-dependent mechanics of receptor activation provides valuable insights into the function of GM-CSF as well as the related cytokines, interleukin-3 and interleukin-5. This knowledge provides opportunities for the development of new therapies to block the action of these cytokines in hematological malignancy and chronic inflammation.

Cutting edge: basophils are transiently recruited into the draining lymph nodes during helminth infection via IL-3, but infection-induced Th2 immunity can develop without basophil lymph node recruitment or IL-3.

Basophils are recognized as immune modulators through their ability to produce IL-4, a key cytokine required for Th2 immunity. It has also recently been reported that basophils are transiently recruited into the draining lymph node (LN) after allergen immunization and that the recruited basophils promote the differentiation of naive CD4 T cells into Th2 effector cells. Using IL-3(-/-) and IL-3Rbeta(-/-) mice, we report in this study that the IL-3/IL-3R system is absolutely required to recruit circulating basophils into the draining LN following helminth infection. Unexpectedly, the absence of IL-3 or of basophil LN recruitment played little role in helminth-induced Th2 immune responses. Moreover, basophil depletion in infected mice did not diminish the development of IL-4-producing CD4 T cells. Our results reveal a previously unknown role of IL-3 in recruiting basophils to the LN and demonstrate that basophils are not necessarily associated with the development of Th2 immunity during parasite infection.

Targeting the Human ßc Receptor Inhibits Contact Dermatitis in a Transgenic Mouse Model.

Allergic contact dermatitis (ACD) is a prevalent and poorly controlled inflammatory disease caused by skin infiltration of T cells and granulocytes. The beta common (ßc) cytokines GM-CSF, IL-3, and IL-5 are powerful regulators of granulocyte function that signal through their common receptor subunit ßc, a property that has made ßc an attractive target to simultaneously inhibit these cytokines. However, the species specificity of ßc has precluded testing of inhibitors of human ßc in mouse models. To overcome this problem, we developed a human ßc receptor transgenic mouse strain with a hematopoietic cell‒specific expression of human ßc instead of mouse ßc. Human ßc receptor transgenic cells responded to mouse GM-CSF and IL-5 but not to IL-3 in vitro and developed tissue pathology and cellular inflammation comparable with those in wild-type mice in a model of ACD. Similarly, Il3-/- mice developed ACD pathology comparable with that of wild-type mice. Importantly, the blocking anti-human ßc antibody CSL311 strongly suppressed ear pinna thickening and histopathological changes typical of ACD and reduced accumulation of neutrophils, mast cells, and eosinophils in the skin. These results show that GM-CSF and IL-5 but not IL-3 are major mediators of ACD and define the human ßc receptor transgenic mouse as a unique platform to test the inhibitors of ßc in vivo.

Sphingosine kinase regulates the rate of endothelial progenitor cell differentiation.

Circulating endothelial progenitor cells (EPCs) are incorporated into foci of neovascularization where they undergo differentiation to mature endothelial cells (ECs). We show here that the enzyme sphingosine kinase-1 (SK-1) regulates the rate and direction of EPC differentiation without effect on the hematopoietic compartment. EPCs have high levels of SK-1 activity, which diminishes with differentiation and is, at least partially, responsible for maintaining their EPC phenotype. EPCs from SK-1 knockout mice form more adherent EC units and acquire a mature EC phenotype more rapidly. Conversely, EPCs from mice overexpressing SK-1 in the EC compartment are retarded in their differentiation. Exogenous regulation of SK-1 levels in normal EPCs, by genetic and pharmacologic means, including the immunomodulating drug FTY720, recapitulates these effects on EC differentiation. SK-1 knockout mice have higher levels of circulating EPCs, an exaggerated response to erythropoietin-induced EPC mobilization, and, in a mouse model of kidney ischemia reperfusion injury, exhibit a recovery similar to that of ischemic mice administered exogenous EPCs. Thus, SK-1 is a critical player in EPC differentiation into EC pointing to the potential utility of SK-1 modifying agents in the specific manipulation of endothelial development and repair.

Molecular basis of cytokine receptor activation.

Cytokines are secreted soluble peptides that precisely regulate multiple cellular functions. Amongst these the GM-CSF/IL-3/IL-5 family of cytokines controls whether hematopoietic cells will survive or apoptose, proliferate, differentiate, migrate, or perform effector functions such as phagocytosis or reactive oxygen species release. Their potent and pleiotropic activities are mediated through binding to high affinity membrane receptors at surprisingly low numbers per cell. Receptor binding triggers a cascade of intracellular signaling events, including reversible phosphorylation of receptor subunits and associated signaling molecules, leading to multiple biological responses, with the prevention of apoptosis or "cell survival" being a key cellular function that underpins all others. Many chronic inflammatory diseases and a number of haematological malignancies are driven by deregulated GM-CSF, IL-3, or IL-5 cytokine receptor signaling, highlighting their importance in disease. A major step in understanding how these cytokine receptors function is to elucidate their three dimensional structure and to relate this to the many signaling pathways emanating from their receptors. We have recently solved the structure of the human GM-CSF receptor complexed to GM-CSF which revealed distinct forms of receptor assembly: a hexamer that comprises two molecules each of GM-CSF, GM-CSF receptor alpha chain and GM-CSF receptor beta chain; and an unexpected dodecamer in which two hexameric complexes associate through a novel site 4. This latter form is necessary to bring JAK2 molecules sufficiently close together to enable full receptor activation. In this review we focus on the most recent insights in cytokine receptor signaling, and in receptor assembly. The stage is now set to link distinct forms of cytokine receptor assembled structures to specific forms of cytokine receptor signaling and function. Armed with this knowledge it may be possible to map distinct cytokine receptor signaling pathways from the cell surface to the cell nucleus which may themselves become new therapeutic targets.

Author Correction: EPO does not promote interaction between the erythropoietin and beta-common receptors.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

EPO does not promote interaction between the erythropoietin and beta-common receptors.

A direct interaction between the erythropoietin (EPOR) and the beta-common (ßc) receptors to form an Innate Repair Receptor (IRR) is controversial. On one hand, studies have shown a functional link between EPOR and ßc receptor in tissue protection while others have shown no involvement of the ßc receptor in tissue repair. To date there is no biophysical evidence to confirm a direct association of the two receptors either in vitro or in vivo. We investigated the existence of an interaction between the extracellular regions of EPOR and the ßc receptor in silico and in vitro (either in the presence or absence of EPO or EPO-derived peptide ARA290). Although a possible interaction between EPOR and ßc was suggested by our computational and genomic studies, our in vitro biophysical analysis demonstrates that the extracellular regions of the two receptors do not specifically associate. We also explored the involvement of the ßc receptor gene (Csf2rb) under anaemic stress conditions and found no requirement for the ßc receptor in mice. In light of these studies, we conclude that the extracellular regions of the EPOR and the ßc receptor do not directly interact and that the IRR is not involved in anaemic stress.

Role of the ß Common (ßc) Family of Cytokines in Health and Disease.

The ß common ([ßc]/CD131) family of cytokines comprises granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5, all of which use ßc as their key signaling receptor subunit. This is a prototypic signaling subunit-sharing cytokine family that has unveiled many biological paradigms and structural principles applicable to the IL-2, IL-4, and IL-6 receptor families, all of which also share one or more signaling subunits. Originally identified for their functions in the hematopoietic system, the ßc cytokines are now known to be truly pleiotropic, impacting on multiple cell types, organs, and biological systems, and thereby controlling the balance between health and disease. This review will focus on the emerging biological roles for the ßc cytokines, our progress toward understanding the mechanisms of receptor assembly and signaling, and the application of this knowledge to develop exciting new therapeutic approaches against human disease.

miR-155 as a potential target of IL-3 signaling in primary AML cells.

miR-155 has emerged as one of the key microRNAs (miRNAs) involved in normal and malignant myelopoiesis, and high expression of this miRNA has been flagged as a strong independent prognostic marker in Acute Myeloid Leukemia (AML). While elevated expression of miR-155 has been associated with FLT3-ITD mutations, other mechanisms which may regulate expression of this miRNA in AML remain largely unknown. Here, we present new evidence that miR-155 may be a prime target of IL-3 signaling in primary AML cells. This finding, together with the increasingly apparent role for miR-155 in oncogenesis, and the upregulation of the IL-3 receptor alpha subunit in AML, lead us to propose this pathway may significantly contribute to the leukemic transformation.

High CD123 levels enhance proliferation in response to IL-3, but reduce chemotaxis by downregulating CXCR4 expression.

High expression of the α chain of the interleukin-3 receptor (IL-3Rα; CD123) is a hallmark of acute myeloid leukemia (AML) leukemic stem cells (LSCs). Elevated CD123 expression is part of the diagnostic immunophenotyping of myeloid leukemia, and higher expression is associated with poor prognosis. However, the biological basis of the poorer prognosis is unclear, and may include heightened IL-3 signaling and non-cell autonomous interactions with the bone marrow (BM) microenvironment. We used TF-1 cells expressing different levels of CD123 and found elevated CD123 levels amplified the proliferative response to exogenous IL-3 and maintained viability in reducing IL-3 concentrations. This was associated with stronger activation of STAT5, Akt, and extracellular signal-regulated kinase 1/2 in vitro. Surprisingly, in vivo e14.5 fetal liver cells transduced with retroviral constructs to express high CD123 failed to engraft in syngeneic recipients. In exploring the underlying mechanism for this, we found that CXCR4, a key molecule involved in LSC/BM interactions, was specifically downregulated in CD123 overexpressing cells in a manner dependent on IL-3 signaling. CXCR4 downregulation was sufficient to alter the chemotactic response of hematopoietic cells to stromal derived factor-1 (SDF-1). Thus, we propose that the overexpression of CD123 in AML LSC dictates their location by altering CXCR4/SDF-1 interaction in the BM, raising the possibility that this mechanism underpins the egress of BM AML LSC and more mature cells into the circulation.

Corrigendum: 14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function.

This corrects the article DOI: 10.1038/ncomms12862.

MicroRNA-194 Promotes Prostate Cancer Metastasis by Inhibiting SOCS2.

Serum levels of miR-194 have been reported to predict prostate cancer recurrence after surgery, but its functional contributions to this disease have not been studied. Herein, it is demonstrated that miR-194 is a driver of prostate cancer metastasis. Prostate tissue levels of miR-194 were associated with disease aggressiveness and poor outcome. Ectopic delivery of miR-194 stimulated migration, invasion, and epithelial-mesenchymal transition in human prostate cancer cell lines, and stable overexpression of miR-194 enhanced metastasis of intravenous and intraprostatic tumor xenografts. Conversely, inhibition of miR-194 activity suppressed the invasive capacity of prostate cancer cell lines in vitro and in vivo Mechanistic investigations identified the ubiquitin ligase suppressor of cytokine signaling 2 (SOCS2) as a direct, biologically relevant target of miR-194 in prostate cancer. Low levels of SOCS2 correlated strongly with disease recurrence and metastasis in clinical specimens. SOCS2 downregulation recapitulated miR-194-driven metastatic phenotypes, whereas overexpression of a nontargetable SOCS2 reduced miR-194-stimulated invasion. Targeting of SOCS2 by miR-194 resulted in derepression of the oncogenic kinases FLT3 and JAK2, leading to enhanced ERK and STAT3 signaling. Pharmacologic inhibition of ERK and JAK/STAT pathways reversed miR-194-driven phenotypes. The GATA2 transcription factor was identified as an upstream regulator of miR-194, consistent with a strong concordance between GATA2 and miR-194 levels in clinical specimens. Overall, these results offer new insights into the molecular mechanisms of metastatic progression in prostate cancer. Cancer Res; 77(4); 1021-34. ©2016 AACR.